I. PROJECT SUMMARY Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic lung infections in people with cystic fibrosis (CF). In the lungs of people with CF, P. aeruginosa changes from a nonmucoid to a mucoid phenotype characterized by the overproduction of alginate. Mucoid conversion is associated with a decrease in lung function, increased patient mortality, and a worsening prognosis of disease, making this phenomenon of great importance to study. Mechanistically, mucoid conversion occurs when truncating mutations in the anti- sigma factor mucA allow for constitutive activity of the sigma factor AlgT, which initiates transcription of the alginate biosynthesis operon as well as other genes of the AlgT regulon. In addition to the overproduction of alginate, mucoid strains isolated from chronic infections often do not express O antigen (OAg). OAg is attached to the distal portion of the lipopolysaccharide molecule and is further subcategorized as long or very long (VL). Each length is under control of a different chain length regulator protein; Wzz1 controls the assembly of long OAg while Wzz2 controls the assembly of VL OAg. Our laboratory has observed that alginate and VL OAg biosynthesis are inversely regulated. Preliminary data I have generated observed that a strain with a mutation that inactivates MucA, resulting in the overproduction of alginate, expresses less VL OAg correlating with decreased expression of Wzz2. We tested if alginate production itself was capable of regulating wzz2 in a mucoid strain by deleting algD, the first gene in the alginate biosynthesis pathway. This nonmucoid strain had increased wzz2 expression suggesting a previously unknown role for the alginate biosynthesis operon in the regulation of VL OAg. Additional preliminary data suggests that nonmucoid cells, when co-cultured with a mucoid strain, express less wzz2. To our knowledge, this is the first example of gene regulation between mucoid and nonmucoid cells within a population. Knowing the steps involved in the regulation of wzz2 and VL OAg at a single-cell and population level will further our understanding of phenotypes associated with mucoid conversion and provide more opportunities for targeted intervention at early stages of infection. The long-term goal of this proposal is to identify how mucoid strains regulate their OAg because interfering with this regulation may destabilize mucoid strains, rendering them unable to establish a chronic infection. The aims of this study are to 1) determine the role of alginate and transcriptional regulators on the expression of wzz2 and 2) determine how wzz2 is repressed in a co-culture and the single-cell dynamics of this regulation. The objective of this proposal is to identify the pathway through which alginate is performing its regulation and to identify novel regulators of wzz2. We will perform a combination of DNA and RNA molecular techniques, protein interaction studies, co-culturing experiments, and single-cell microscopy. By interfering with the establishment of a chronic P. aeruginosa infection we can provide a better outcome for people with CF.